Nucleoside derivatives as inhibitors of rna-dependent rna viral polymerase

ABSTRACT

Thee present invention provides nucleoside compounds and certain derivatives thereof which are inhibitors of RNA-dependent RNA viral polymerase. These compounds are inhibitors of RNA-dependent RNA via replication and are useful for the treatment of RNA-dependent RNA viral infection. They are particularly useful as inhibitors of hepatitis C virus (HCV) NS5B polymerase, as inhibitors of HCV replication, and/or for the treatment of hepatitis C infection. The invention also describes pharmaceutical compositions containing such nucleoside compounds alone or in combination with other agents active against RNA-dependent RNA viral infection, in particular HCV infection. Also disclosed are methods of inhibiting RNA-dependent RNA polymerase, inhibiting RNA-dependent RNA viral replication, and/or creating RNA-dependent RNA viral infection with the nucleoside compounds of the present invention.

FIELD OF THE INVENTION

The present invention is concerned with nucleoside compounds and certainderivatives thereof, their synthesis, and their use as inhibitors ofRNA-dependent RNA viral polymerase. The compounds of the presentinvention are inhibitors of RNA-dependent RNA viral replication and areuseful for the treatment of RNA-dependent RNA viral infection. They areparticularly useful as inhibitors of hepatitis C virus (HCV) NS5Bpolymerase, as inhibitors of HCV replication, and for the treatment ofhepatitis C infection.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) infection is a major health problem that leadsto chronic liver disease, such as cirrhosis and hepatocellularcarcinoma, in a substantial number of infected individuals, estimated tobe 2-15% of the world's population. There are an estimated 4.5 millioninfected people in the United States alone, according to the U.S. Centerfor Disease Control. According to the World Health Organization, thereare more than 200 million infected individuals worldwide, with at least3 to 4 million people being infected each year. Once infected, about 20%of people clear the virus, but the rest harbor HCV the rest of theirlives. Ten to twenty percent of chronically infected individualseventually develop liver-destroying cirrhosis or cancer. The viraldisease is transmitted parenterally by contaminated blood and bloodproducts, contaminated needles, or sexually and vertically from infectedmothers or carrier mothers to their off-spring. Current treatments forHCV infection, which are restricted to immunotherapy with recombinantinterferon-α alone or in combination with the nucleoside analogribavirin, are of limited clinical benefit. Moreover, there is noestablished vaccine for HCV. Consequently, there is an urgent need forimproved therapeutic agents that effectively combat chronic HCVinfection. The state of the art in the treatment of HCV infection hasbeen reviewed, and reference is made to the following publications: B.Dymock, et al., “Novel approaches to the treatment of hepatitis C virusinfection,” Antiviral Chemistry & Chemotherapy, 11: 79-96 (2000); H.Rosen, et al., “Hepatitis C virus: current understanding and prospectsfor future therapies,” Molecular Medicine Today, 5: 393-399 (1999); D.Moradpour, et al., “Current and evolving therapies for hepatitis C,”European J. Gastroenterol. Hepatol., 11: 1189-1202 (1999); R.Bartenschlager, “Candidate Targets for Hepatitis C Virus-SpecificAntiviral Therapy,” Intervirology, 40: 378-393 (1997); G. M. Lauer andB. D. Walker, “Hepatitis C Virus Infection,” N. Engl. J. Med., 345:41-52 (2001); B. W. Dymock, “Emerging therapies for hepatitis C virusinfection,” Emerging Drugs, 6: 13-42 (2001); and C. Crabb, “Hard-WonAdvances Spark Excitement about Hepatitis C,” Science: 506-507 (2001);the contents of all of which are incorporated by reference herein intheir entirety.

Different approaches to HCV therapy have been taken, which include theinhibition of viral serine proteinase (NS3 protease), helicase, andRNA-dependent RNA polymerase (NS5B), and the development of a vaccine.

The HCV virion is an enveloped positive-strand RNA virus with a singleoligoribonucleotide genomic sequence of about 9600 bases which encodes apolyprotein of about 3,010 amino acids. The protein products of the HCVgene consist of the structural proteins C, E1, and E2, and thenon-structural proteins NS2, NS3, NS4A and NS4B, and NS5A and NS5B. Thenonstructural (NS) proteins are believed to provide the catalyticmachinery for viral replication. The NS3 protease releases NS5B, theRNA-dependent RNA polymerase from the polyprotein chain. HCV NS5Bpolymerase is required for the synthesis of a double-stranded RNA from asingle-stranded viral RNA that serves as a template in the replicationcycle of HCV. NS5B polymerase is therefore considered to be an essentialcomponent in the HCV replication complex [see K. Ishi, et al.,“Expression of Hepatitis C Virus NS5B Protein: Characterization of ItsRNA Polymerase Activity and RNA Binding,” Hepatology, 29: 1227-1235(1999) and V. Lohmann, et al., “Biochemical and Kinetic Analyses of NS5BRNA-Dependent RNA Polymerase of the Hepatitis C Virus,” Virology, 249:108-118 (1998)]. Inhibition of HCV NS5B polymerase prevents formation ofthe double-stranded HCV RNA and therefore constitutes an attractiveapproach to the development of HCV-specific antiviral therapies.

It has now been found that nucleoside compounds of the present inventionand certain derivatives thereof are potent inhibitors of RNA-dependentRNA viral replication and in particular HCV replication. The5′-triphosphate derivatives of these nucleoside compounds are inhibitorsof RNA-dependent RNA viral polymerase and in particular HCV NS5Bpolymerase. The instant nucleoside compounds and derivatives thereof areuseful to treat RNA-dependent RNA viral infection and in particular HCVinfection.

It is therefore an object of the present invention to provide nucleosidecompounds and certain derivatives thereof which are useful as inhibitorsof RNA-dependent RNA viral polymerase and in particular as inhibitors ofHCV NS5B polymerase.

It is another object of the present invention to provide nucleosidecompounds and certain derivatives thereof which are useful as inhibitorsof the replication of an RNA-dependent RNA virus and in particular asinhibitors of the replication of hepatitis C virus.

It is another object of the present invention to provide nucleosidecompounds and certain derivatives thereof which are useful in thetreatment of RNA-dependent RNA viral infection and in particular in thetreatment of HCV infection.

It is another object of the present invention to provide pharmaceuticalcompositions comprising the nucleoside compounds of the presentinvention in association with a pharmaceutically acceptable carrier.

It is another object of the present invention to provide pharmaceuticalcompositions comprising the nucleoside compounds and derivatives thereofof the present invention for use as inhibitors of RNA-dependent RNAviral polymerase and in particular as inhibitors of HCV NS5B polymerase.

It is another object of the present invention to provide pharmaceuticalcompositions comprising the nucleoside compounds and derivatives thereofof the present invention for use as inhibitors of RNA-dependent RNAviral replication and in particular as inhibitors of HCV replication.

It is another object of the present invention to provide pharmaceuticalcompositions comprising the nucleoside compounds and derivatives thereofof the present invention for use in the treatment of RNA-dependent RNAviral infection and in particular in the treatment of HCV infection.

It is another object of the present invention to provide pharmaceuticalcompositions comprising the nucleoside compounds and derivatives thereofof the present invention in combination with other agents active againstan RNA-dependent RNA virus and in particular against HCV.

It is another object of the present invention to provide methods for theinhibition of RNA-dependent RNA viral polymerase and in particular forthe inhibition of HCV NS5B polymerase.

It is another object of the present invention to provide methods for theinhibition of RNA-dependent RNA viral replication and in particular forthe inhibition of HCV replication.

It is another object of the present invention to provide methods for thetreatment of RNA-dependent RNA viral infection and in particular for thetreatment of HCV infection.

It is another object of the present invention to provide methods for thetreatment of RNA-dependent RNA viral infection in combination with otheragents active against RNA-dependent RNA virus and in particular for thetreatment of HCV infection in combination with other agents activeagainst HCV.

It is another object of the present invention to provide nucleosidecompounds and certain derivatives thereof and their pharmaceuticalcompositions for use as a medicament for the inhibition of RNA-dependentRNA viral replication and/or the treatment of RNA-dependent RNA viralinfection and in particular for the inhibition of HCV replication and/orthe treatment of HCV infection.

It is another object of the present invention to provide for the use ofthe nucleoside compounds and certain derivatives thereof of the presentinvention and their pharmaceutical compositions for the manufacture of amedicament for the inhibition of RNA-dependent RNA viral replicationand/or the treatment of RNA-dependent RNA viral infection and inparticular for the inhibition of HCV replication and/or the treatment ofHCV infection.

These and other objects will become readily apparent from the detaileddescription which follows.

SUMMARY OF THE INVENTION

The present invention relates to compounds of structural formula I ofthe indicated stereochemical configuration:

or a pharmaceutically acceptable salt thereof;

wherein R¹ is C₁₋₄ alkyl, wherein alkyl is unsubstituted or substitutedwith hydroxy, amino, C₁₋₄ alkoxy, C₁₋₄ alkylthio, or one to threefluorine atoms;

R² is amino, fluorine, hydroxy, C₁₋₁₀ alkylcarbonyloxy, mercapto, orC₁₋₄ alkoxy;

R³ and R⁴ are each independently hydrogen, C₁₋₁₆ alkylcarbonyl, C₂₋₁₈alkenylcarbonyl, C₁₋₁₀ alkyloxycarbonyl, C₃₋₆ cycloalkylcarbonyl, C₃₋₆cycloalkyloxycarbonyl, CH₂O(C═O)C₁₋₄ alkyl, CH(C₁₋₄alkyl)O(C═O)C₁₋₄alkyl, or an amino acyl residue of structural formula

with the proviso that at least one of R³ and R⁴ is not hydrogen;

R⁵ and R⁶ are each independently hydrogen, methyl, hydroxymethyl, orfluoromethyl;

R⁷ is hydrogen, C₁₋₄ alkyl, C₂₋₄ alkynyl, halogen, cyano, carboxy, C₁₋₄alkyloxycarbonyl, azido, amino, C₁₋₄ alkylamino, di(C₁₋₄ alkyl)amino,hydroxy, C₁₋₆ alkoxy, C₁₋₆ alkylthio, C₁₋₆ alkylsulfonyl, or (C₁₋₄alkyl)₀₋₂ aminomethyl;

R⁸ is hydrogen, cyano, nitro, C₁₋₃ alkyl, NHCONH₂, CONR¹¹R¹¹, CSNR₁₁R¹¹,COOR¹¹, C(═NH)NH₂, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₄ alkylamino, di(C₁₋₄alkyl)amino, halogen, (1,3-oxazol-2-yl), (1,3-thiazol-2-yl), or(imidazol-2-yl); wherein alkyl is unsubstituted or substituted with oneto three groups independently selected from halogen, amino, hydroxy,carboxy, and C₁₋₃ alkoxy;

R⁹ is hydrogen, hydroxy, mercapto, halogen, C₁₋₄ alkoxy, C₁₋₄ alkylthio,C₁₋₈ alkylcarbonyloxy, C₃₋₆ cycloalkylcarbonyloxy, C₁₋₈alkyloxycarbonyloxy, C₃₋₆ cycloalkyloxycarbonyloxy, OCH₂CH₂SC(═O)C₁₋₄alkyl, OCH₂O(C═O)C₁₋₄ alkyl, OCH(C₁₋₄ alkyl)O(C═O)C₁₋₄ alkyl, amino,C₁₋₄ alkylamino, di(C₁₋₄ alkyl)amino, C₃₋₆ cycloalkylamino, or di(C₃₋₆cycloalkyl)amino;

R¹⁰ is hydrogen, hydroxy, halogen, C₁₋₄ alkoxy, amino, C₁₋₄ alkylamino,di(C₁₋₄alkyl)amino, C₃₋₆ cycloalkylamino, or di(C₃₋₆ cycloalkylarnino);

each R¹¹ is independently hydrogen or C₁₋₆ alkyl;

R¹² is hydrogen, C₁₋₄ alkyl, or phenyl C₀₋₂ alky; and

R¹³ is hydrogen, C₁₋₄ alkyl, C₁₋ ₄ acyl, benzoyl, C₁₋₄ alkyloxycarbonyl,phenyl C₀₋₂ alkyloxycarbonyl, C₁₋₄ alkylaminocarbonyl, phenyl C₀₋₂alkylaminocarbonyl, C₁₋₄ alkylsulfonyl, or phenyl C₀₋₂ alkylsulfonyl.

The compounds of formula I are useful as inhibitors of RNA-dependent RNAviral polymerase and in particular of HCV NS5B polymerase. They are alsoinhibitors of RNA-dependent RNA viral replication and in particular ofHCV replication and are useful for the treatment of RNA-dependent RNAviral infection and in particular for the treatment of HCV infection.

Also encompassed within the present invention are pharmaceuticalcompositions containing the compounds alone or in combination with otheragents active against RNA-dependent RNA virus and in particular againstHCV as well as methods for the inhibition of RNA-dependent RNA viralreplication and for the treatment of RNA-dependent RNA viral infection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds of structural formula I ofthe indicated stereochemical configuration:

or a pharmaceutically acceptable salt thereof;

wherein R¹ is C₁₋₄ alkyl, wherein alkyl is unsubstituted or substitutedwith hydroxy, amino, C₁₋₄ alkoxy, C₁₋₄ alkylthio, or one to threefluorine atoms;

R² is amino, fluorine, hydroxy, C₁₋₁₀ alkylcarbonyloxy, mercapto, orC₁₋₄ alkoxy;

R³ and R⁴ are each independently hydrogen, C₁₋₁₆ alkylcarbonyl, C₂₋₁₈alkenylcarbonyl, C₁₋₁₀ alkyloxycarbonyl, C₃₋₆ cycloalkylcarbonyl, C₃₋₆cycloalkyloxycarbonyl, CH₂O(C═O)C₁₋₄ alkyl, CH(C₁₋₄ alkyl)O(C═O)C₁₋₄alkyl, or an amino acyl residue of structural formula

with the proviso that at least one of R³ and R⁴ is not hydrogen;

R⁵ and R⁶ are each independently hydrogen, methyl, hydroxymethyl, orfluoromethyl;

R⁷ is hydrogen, C₁₋₄ alkyl, C₂₋₄ alkynyl, halogen, cyano, carboxy, C₁₋₄alkyloxycarbonyl, azido, amino, C₁₋₄ alkylamino, di(C₁₋₄ alkyl)amino,hydroxy, C₁₋₆ alkoxy, C₁₋₆ alkylthio, C₁₋₆ alkylsulfonyl, or (C₁₋₄alkyl)₀₋₂ aminomethyl;

R⁸ is hydrogen, cyano, nitro, C₁₋₃ alkyl, NHCONH₂, CONR¹¹R¹¹, CSNR¹¹R¹¹,COOR¹¹, C(═NH)NH₂, hydroxy, C₁₋₃ alkoxy, amino, C₁₋₄ alkylamino, di(C₁₋₄alkyl)amino, halogen, (1,3-oxazol-2-yl), (1,3-thiazol-2-yl), or(imidazol-2-yl); wherein alkyl is unsubstituted or substituted with oneto three groups independently selected from halogen, amino, hydroxy,carboxy, and C₁₋₃ alkoxy;

R⁹ is hydrogen, hydroxy, mercapto, halogen, C₁₋₄ alkoxy, C₁₋₄ alkylthio,C₁₋₈ alkylcarbonyloxy, C₃₋₆ cycloalkylcarbonyloxy, C₁₋₈alkyloxycarbonyloxy, C₃₋₆ cycloalkyloxycarbonyloxy, OCH₂CH₂SC(═O)C₁₋₄alkyl, OCH₂O(C═O)C₁₋₄ alkyl, OCH(C₁₋₄ alkyl)O(C═O)C₁₋₄ alkyl, amino,C₁₋₄ alkylamino, di(C₁₋₄ alkyl)amino, C₃₋₆ cycloalkylamino, or di(C₃₋₆cycloalkyl)amino;

R¹⁰ is hydrogen, hydroxy, halogen, C₁₋₄ alkoxy, amino, C₁₋₄ alkylamino,di(C₁₋₄ alkyl)amino, C₃₋₆ cycloalkylamino, or di(C₃₋₆ cycloalkylamino);

each R¹¹ is independently hydrogen or C₁₋₆ alkyl;

R¹² is hydrogen, C₁₋₄ alkyl, or phenyl C₀₋₂ alkyl; and

R¹³ is hydrogen, C₁₋₄ alkyl, C₁₋₄ acyl, benzoyl, C₁₋₄ alkyloxycarbonyl,phenyl C₀₋₂ alkyloxycarbonyl, C₁₋₄ alkylaminocarbonyl, phenyl C₀₋₂alkylaminocarbonyl, C₁₋₄ alkylsulfonyl, or phenyl C₀₋₂ alkylsulfonyl.

The compounds of formula I are useful as inhibitors of RNA-dependent RNAviral polymerase. They are also inhibitors of RNA-dependent RNA viralreplication and are useful for the treatment of RNA-dependent RNA viralinfection.

In one embodiment of the compounds of structural formula I are thecompounds of structural formula II:

or a pharmaceutically acceptable salt thereof;wherein

R¹ is C₁₋₃ alkyl, wherein alkyl is unsubstituted or substituted withhydroxy, amino, C₁₋₃ alkoxy, C₁₋₃ alkylthio, or one to three fluorineatoms;

R² is hydroxy, amino, fluoro, or C₁₋₃ alkoxy;

R³ and R⁴ are each independently hydrogen, C₁₋₈ alkylcarbonyl, or C₃₋₆cycloalkylcarbonyl, with the proviso that at least one of R³ and R⁴ isnot hydrogen;

R⁷ is hydrogen, amino, or C₁₋₄ alkylamino;

R⁸ is hydrogen, cyano, methyl, halogen, or CONH₂; and

R⁹ and R¹⁰ are each independently hydrogen, halogen, hydroxy, or amino.

In a second embodiment of the compounds of structural formula I are, thecompounds of structural formula II wherein:

R¹ is methyl, fluoromethyl, hydroxymethyl, difluoromethyl,trifluoromethyl, or aminomethyl;

R² is hydroxy, amino, fluoro, or methoxy;

R³ and R⁴ are each independently hydrogen or C₁₋₈ alkylcarbonyl, withthe proviso that at least one of R³ and R⁴ is not hydrogen;

R⁷ is hydrogen or amino;

R⁸ is hydrogen, cyano, methyl, halogen, or CONH₂; and

R⁹ and R¹⁰ are each independently hydrogen, fluoro, hydroxy, or amino.

Illustrative, but nonlimiting, examples of compounds of the presentinvention of structural formula I which are useful as inhibitors ofRNA-dependent RNA viral polymerase are the following:

-   4-amino-7-[2-C-methyl-3,5-di-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine;-   4-amino-7-[2-C-methyl-3-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine;-   4-amino-7-[2-C-methyl-5-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine;    and-   4-amino-7-[2-C-methyl-2,3,5-tri-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine;    or a pharmaceutically acceptable salt thereof.

In one embodiment of the present invention, the nucleoside compounds ofthe present invention are useful as inhibitors of positive-sensesingle-stranded RNA-dependent RNA viral polymerase, inhibitors ofpositive-sense single-stranded RNA-dependent RNA viral replication,and/or for the treatment of positive-sense single-stranded RNA-dependentRNA viral infection. In a class of this embodiment, the positive-sensesingle-stranded RNA-dependent RNA virus is a Flaviviridae virus or aPicornaviridae virus. In a subclass of this class, the Picornaviridaevirus is a rhinovirus, a poliovirus, or a hepatitis A virus. In a secondsubclass of this class, the Flaviviridae virus is selected from thegroup consisting of hepatitis C virus, yellow fever virus, dengue virus,West Nile virus, Japanese encephalitis virus, Banzi virus, and bovineviral diarrhea virus (BVDV). In a subclass of this subclass, theFlaviviridae virus is hepatitis C virus.

Another aspect of the present invention is concerned with a method forinhibiting RNA-dependent RNA viral polymerase, a method for inhibitingRNA-dependent RNA viral replication, and/or a method for treatingRNA-dependent RNA viral infection in a mammal in need thereof comprisingadministering to the mammal a therapeutically effective amount of acompound of structural formula I.

In one embodiment of this aspect of the present invention, theRNA-dependent RNA viral polymerase is a positive-sense single-strandedRNA-dependent RNA viral polymerase. In a class of this embodiment, thepositive-sense single-stranded RNA-dependent RNA viral polymerase is aFlaviviridae viral polymerase or a Picornaviridae viral polymerase. In asubclass of this class, the Picornaviridae viral polymerase isrhinovirus polymerase, poliovirus polymerase, or hepatitis A viruspolymerase. In a second subclass of this class, the Flaviviridae viralpolymerase is selected from the group consisting of hepatitis C viruspolymerase, yellow fever virus polymerase, dengue virus polymerase, WestNile virus polymerase, Japanese encephalitis virus polymerase, Banzivirus polymerase, and bovine viral diarrhea virus (BVDV) polymerase. Ina subclass of this subclass, the Flaviviridae viral polymerase ishepatitis C virus polymerase.

In a second embodiment of this aspect of the present invention, theRNA-dependent RNA viral replication is a positive-sense single-strandedRNA-dependent RNA viral replication. In a class of this embodiment, thepositive-sense single-stranded RNA-dependent RNA viral replication isFlaviviridae viral replication or Picornaviridae viral replication. In asubclass of this class, the Picornaviridae viral replication isrhinovirus replication, poliovirus replication, or hepatitis A virusreplication. In a second subclass of this class, the Flaviviridae viralreplication is selected from the group consisting of hepatitis C virusreplication, yellow fever virus replication, dengue virus replication,West Nile virus replication, Japanese encephalitis virus replication,Banzi virus replication, and bovine viral diarrhea virus replication. Ina subclass of this subclass, the Flaviviridae viral replication ishepatitis C virus replication.

In a third embodiment of this aspect of the present invention, theRNA-dependent RNA viral infection is a positive-sense single-strandedRNA-dependent viral infection. In a class of this embodiment, thepositive-sense single-stranded RNA-dependent RNA viral infection isFlaviviridae viral infection or Picornaviridae viral infection. In asubclass of this class, the Picornaviridae viral infection is rhinovirusinfection, poliovirus infection, or hepatitis A virus infection. In asecond subclass of this class, the Flaviviridae viral infection isselected from the group consisting of hepatitis C virus infection,yellow fever virus infection, dengue virus infection, West Nile virusinfection, Japanese encephalitis virus infection, Banzi virus infection,and bovine viral diarrhea virus infection. In a subclass of thissubclass, the Flaviviridae viral infection is hepatitis C virusinfection.

Throughout the instant application, the following terms have theindicated meanings:

The alkyl groups specified above are intended to include those alkylgroups of the designated length in either a straight or branchedconfiguration. Exemplary of such alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl,isohexyl, and the like.

The term “alkenyl” shall mean straight or branched chain alkenes of twoto six total carbon atoms, or any number within this range (e.g.,ethenyl, propenyl, butenyl, pentenyl, etc.).

The term “alkynyl” shall mean straight or branched chain alknes of twoto six total carbon atoms, or any number within this range (e.g.,ethynyl, propynyl, butynyl, pentynyl, etc.).

The term “cycloalkyl” shall mean cyclic rings of alkanes of three toeight total carbon atoms, or any number within this range (i.e.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, orcyclooctyl).

The term “cycloheteroalkyl” is intended to include non-aromaticheterocycles containing one or two heteroatoms selected from nitrogen,oxygen and sulfur. Examples of 4-6-membered cycloheteroalkyl includeazetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, thiamorpholinyl,imidazolidinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydrothiophenyl, piperazinyl, and the like.

The term “alkoxy” refers to straight or branched chain alkoxides of thenumber of carbon atoms specified (e.g., C₁₋₄ alkoxy), or any numberwithin this range [i.e., methoxy (MeO—), ethoxy, isopropoxy, etc.].

The term “alkylthio” refers to straight or branched chain alkylsulfidesof the number of carbon atoms specified (e.g., C₁₋₄ alkylthio), or anynumber within this range [i.e., methylthio (MeS—), ethylthio,isopropylthio, etc.].

The term “alkylamino” refers to straight or branched alkylamines of thenumber of carbon atoms specified (e.g., C₁₋₄ alkylamino), or any numberwithin this range [i.e., methylamino, ethylamino, isopropylamino,t-butylamino, etc.].

The term “cycloalkylamino” refers to saturated aminohydrocarbonscontaining one ring of the number of carbon atoms specified (e.g., C₃₋₆cycloalkylamino), or any number within this range [i.e.,cyclopropylamino, cyclobutylamino, cyclopentylamino, andcyclohexylamino].

The term “alkylsulfonyl” refers to straight or branched chainalkylsulfones of the number of carbon atoms specified (e.g., C₁₋₆alkylsulfonyl), or any number within this range [i.e., methylsulfonyl(MeSO₂—), ethylsulfonyl, isopropylsulfonyl, etc.].

The term “alkyloxycarbonyl” refers to straight or branched chain estersof a carboxylic acid derivative of the present invention of the numberof carbon atoms specified (e.g., C₁₋₄ alkyloxycarbonyl), or any numberwithin this range [i.e., methyloxycarbonyl (MeOCO—), ethyloxycarbonyl,or butyloxycarbonyl].

The term “alkenylcarbonyl” refers to a straight or branched chainunsaturated alkylcarbonyl group having two to sixteen total carbon atomsand containing one to three double bonds in the chain.

The term “aryl” includes both phenyl, naphthyl, and pyridyl. The arylgroup is optionally substituted with one to three groups independentlyselected from C₁₋₄ alkyl, halogen, cyano, nitro, trifluoromethyl, C₁₋₄alkoxy, and C₁₋₄ alkylthio.

The term “halogen” is intended to include the halogen atoms fluorine,chlorine, bromine and iodine.

The term “substituted” shall be deemed to include multiple degrees ofsubstitution by a named substituent. Where multiple substituent moietiesare disclosed or claimed, the substituted compound can be independentlysubstituted by one or more of the disclosed or claimed substituentmoieties, singly or plurally.

When R¹² in the amino acyl residue shown below is not hydrogen,

the amino acyl residue contains an asymmetric center and is intended toinclude the individual R- and S-enantioners as well as RS-racemicmixtures.

The term “composition”, as in “pharmaceutical composition,” is intendedto encompass a product comprising the active ingredient(s) and the inertI ingredient(s) that make up the carrier, as well as any product whichresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions of the present invention encompass anycomposition made by admixing a compound of the present invention and apharmaceutically acceptable carrier.

The terms “administration of” and “administering a” compound should beunderstood to mean providing a compound of the invention or a prodrug ofa compound of the invention to the individual in need.

Another aspect of the present invention is concerned with a method ofinhibiting HCV NS5B polymerase, inhibiting HCV replication, or treatingHCV infection with a compound of the present invention in combinationwith one or more agents useful for treating HCV infection. Such agentsactive against HCV include, but are not limited to, ribavirin,levovirin, viramidine, thymosin alpha-1, interferon-β, interferon-α,pegylated interferon-α (peginterferon-α), a combination of interferon-αand ribavirin, a combination of peginterferon-α and ribavirin, acombination of interferon-α and levovirin, and a combination ofpeginterferon-α and levovirin. Interferon-α includes, but is not limitedto, recombinant interferon-α2a (such, as Roferon interferon availablefrom Hoffmann-LaRoche, Nutley, N.J.), pegylated interferon-α2a(Pegasys™), interferon-α2b (such as Intron-A interferon available fromSchering Corp., Kenilworth, N.J.), pegylated interferon-α2b(PegIntron™), a recombinant consensus interferon (such as interferonalphacon-1), and a purified interferon-α product. Amgen's recombinantconsensus interferon has the brand name Infergen®. Levovirin is theL-enantiomer of ribavirin which has shown immunomodulatory activitysimilar to ribavirin. Viramidine represents an analog of ribavirindisclosed in WO 01/60379 (assigned to ICN Pharmaceuticals). Inaccordance with this method of the present invention, the individualcomponents of the combination can be administered separately atdifferent times during the course of therapy or concurrently in dividedor single combination forms. The instant invention is therefore to beunderstood as embracing all such regimes of simultaneous or alternatingtreatment, and the term “administering” is to be interpretedaccordingly. It will be understood that the scope of combinations of thecompounds of this invention with other agents useful for treating HCVinfection includes in principle any combination with any pharmaceuticalcomposition for treating HCV infection. When a compound of the presentinvention or a pharmaceutically acceptable salt thereof is used incombination with a second therapeutic agent active against HCV, the doseof each compound may be either the same as or different from the dosewhen the compound is used alone.

For the treatment of HCV infection, the compounds of the presentinvention may also be administered in combination with an agent that isan inhibitor of HCV NS3 serine protease. HCV NS3 serine protease is anessential viral enzyme and has been described to be an excellent targetfor inhibition of HCV replication. Both substrate and non-substratebased inhibitors of HCV NS3 protease inhibitors are disclosed in WO98/22496, WO 98/46630, WO 99/07733, WO 99/07734, WO 99/38888, WO99/50230, WO 99/64442, WO 00/09543, WO 00/59929, and GB-2337262. HCV NS3protease as a target for the development of inhibitors of HCVreplication and for the treatment of HCV infection is discussed in B. W.Dymock, “merging therapies for hepatitis C virus infection,” EmergingDrugs, 6: 13-42 (2001).

Ribavirin, levovirin, and viramidine may exert their anti-HCV effects bymodulating intracellular pools of guanine nucleotides via inhibition ofthe intracellular enzyme inosine monophosphate dehydrogenase (IMPDH).IMPDH is the rate-limiting enzyme on the biosynthetic route in de novoguanine nucleotide biosynthesis. Ribavirin is readily phosphorylatedintracellularly and the monophosphate derivative is an inhibitor ofIMPDH. Thus, inhibition of IMPDH represents another useful target forthe discovery of inhibitors of HCV replication. Therefore, the compoundsof the present invention may also be administered in combination with aninhibitor of IMPDH, such as VX497, which is disclosed in WO 97/41211 andWO 01/00622 (assigned to Vertex); another IMPDH inhibitor, such as thatdisclosed in WO 00/25780 (assigned to Bristol-Myers Squibb); ormycophenolate mofetil [see A. C. Allison and E. M. Eugui, Agents Action,44 (Suppl.): 165 (1993)].

For the treatment of HCV infection, the compounds of the presentinvention may also be administered in combination with the antiviralagent amantadine (1-aminoadamantane) [for a comprehensive description ofthis agent, see J. Kirschbaum, Anal. Profiles Drug Subs. 12: 1-36(1983)].

The compounds of the present invention may also be combined for thetreatment of HCV infection with antiviral 2′-C-branched ribonucleosidesdisclosed in R. E. Harry-O'kuru, et al., J. Org. Chem., 62: 1754-1759(1997); M. S. Wolfe, et al., Tetrahedron Lett., 36: 7611-7614 (1995);U.S. Pat. No. 3,480,613 (Nov. 25, 1969); International PublicationNumber WO 01/90121 (29 Nov. 2001); International Publication Number WO01/92282 (6 Dec. 2001); and International Publication Number WO 02/32920(25 Apr. 2002); the contents of each of which are incorporated byreference in their entirety. Such 2′-C-branched ribonucleosides include,but are not limited to, 2′-C-methyl-cytidine, 2′-C-methyl-uridine,2′-C-methyl-adenosine, 2′-C-methyl-guanosine, and9-(2-C-methyl-β-D-ribofuranosyl)-2,6-diaminopurine.

By “pharmaceutically acceptable” is meant that the carrier, diluent, orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

Also included within the present invention are pharmaceuticalcompositions comprising the nucleoside compounds and derivatives thereofof the present invention in association with a pharmaceuticallyacceptable carrier. Another example of the invention is a pharmaceuticalcomposition made by combining any of the compounds described above and apharmaceutically acceptable carrier. Another illustration of theinvention is a process for making a pharmaceutical compositioncomprising combining any of the compounds described above and apharmaceutically acceptable carrier.

Also included within the present invention are pharmaceuticalcompositions useful for inhibiting RNA-dependent RNA viral polymerase inparticular HCV NS5B polymerase comprising an effective amount of acompound of the present invention and a pharmaceutically acceptablecarrier. Pharmaceutical compositions useful for treating RNA-dependentRNA viral infection in particular HCV infection are also encompassed bythe present invention as well as a method of inhibiting RNA-dependentRNA viral polymerase in particular HCV NS5B polymerase and a method oftreating RNA-dependent viral replication and in particular HCVreplication. Additionally, the present invention is directed to apharmaceutical composition comprising a therapeutically effective amountof a compound of the present invention in combination with atherapeutically effective amount of another agent active againstRNA-dependent RNA virus and in particular against HCV. Agents activeagainst HCV include, but are not limited to, ribavirin, levovirin,viramidine, thymosin alpha-1, an inhibitor of HCV NS3 serine protease,interferon-α, pegylated interferon-α (peginterferon-α), a combination ofinterferon-α and ribavirin, a combination of peginterferon-α andribavirin, a combination of interferon-α and levovirin, and acombination of peginterferon-α and levovirin. Interferon-α includes, butis not limited to, recombinant interferon-α2a (such as Roferoninterferon available from Hoffmann-LaRoche, Nutley, N.J.),interferon-α2b (such as Intron-A interferon available from ScheringCorp., Kenilworth, N.J.), a consensus interferon, and a purifiedinterferon-α product. For a discussion of ribavirin and its activityagainst HCV, see J. O. Saunders and S. A. Raybuck, “InosineMonophosphate Dehydrogenase: Consideration of Structure, Kinetics, andTherapeutic Potential,” Ann. Rep. Med. Chem., 35: 201-210 (2000).

Another aspect of the present invention provides for the use of thenucleoside compounds and derivatives thereof and their pharmaceuticalcompositions for the manufacture of a medicament for the inhibition ofRNA-dependent RNA viral replication, in particular HCV replication,and/or the treatment of RNA-dependent RNA viral infection, in particularHCV infection. Yet a further aspect of the present invention providesfor the nucleoside compounds and derivatives thereof and theirpharmaceutical compositions for use as a medicament for the inhibitionof RNA-dependent RNA viral replication, in particular HCV replication,and/or for the treatment of RNA-dependent RNA viral infection, inparticular HCV infection.

The pharmaceutical compositions of the present invention comprise acompound of structural formula I as an active ingredient or apharmaceutically acceptable salt thereof, and may also contain apharmaceutically acceptable carrier and optionally other therapeuticingredients.

The compositions include compositions suitable for oral, rectal,topical, parenteral (including subcutaneous, intramuscular, andintravenous), ocular (ophthalmic), pulmonary (nasal or buccalinhalation), or nasal administration, although the most suitable routein any given case will depend on the nature and severity of theconditions being treated and on the nature of the active ingredient.They may be conveniently presented in unit dosage form and prepared byany of the methods well-known in the art of pharmacy.

In practical use, the compounds of structural formula I can be combinedas the active ingredient in intimate admixture with a pharmaceuticalcarrier according to conventional pharmaceutical compounding techniques.The carrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral(including intravenous). In preparing the compositions for oral dosageform, any of the usual pharmaceutical media may be employed, such as,for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents and the like in the case of oral liquidpreparations, such as, for example, suspensions, elixirs and solutions;or carriers such as starches, sugars, microcrystalline cellulose,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like in the case of oral solid preparations such as, forexample, powders, hard and soft capsules and tablets, with the solidoral preparations being preferred over the liquid preparations.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit form in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe coated by standard aqueous or nonaqueous techniques. Suchcompositions and preparations should contain at least 0.1 percent ofactive compound. The percentage of active compound in these compositionsmay, of course, be varied and may conveniently be between about 2percent to about 60 percent of the weight of the unit. The amount ofactive compound in such therapeutically useful compositions is such thatan effective dosage will be obtained. The active compounds can also beadministered intranasally as, for example, liquid drops or spray.

The tablets, pills, capsules, and the like may also contain a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,potato starch, alginic acid; a lubricant such as magnesium stearate; anda sweetening agent such as sucrose, lactose or saccharin. When a dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar or both. A syrup or elixir may contain, in additionto the active ingredient, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Compounds of structural formula I may also be administered parenterally.Solutions or suspensions of these active compounds can be prepared inwater suitably mixed with a surfactant such as hydroxy-propylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols and mixtures thereof in, oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g. glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

Any suitable route of administration may be employed for providing amammal, especially a human with an effective dosage of a compound of thepresent invention. For example, oral, rectal, topical, parenteral,ocular, pulmonary, nasal, and the like may be employed. Dosage formsinclude tablets, troches, dispersions, suspensions, solutions, capsules,creams, ointments, aerosols, and the like. Preferably compounds ofstructural formula I are administered orally.

For oral administration to humans, the dosage range is 0.01 to 1000mg/kg body weight in divided doses. In one embodiment the dosage rangeis 0.1 to 100 mg/kg body weight in divided doses. In another embodimentthe dosage range is 0.5 to 20 mg/kg body weight in divided doses. Fororal administration, the compositions are preferably provided in theform of tablets or capsules containing 1.0 to 1000 milligrams of theactive ingredient, particularly, 1, 5, 10, 15, 20, 25, 50, 75, 100, 150,200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 milligrams of theactive ingredient for the symptomatic adjustment of the dosage to thepatient to be treated.

The effective dosage of active ingredient employed may vary depending onthe particular compound employed, the mode of administration, thecondition being treated and the severity of the condition being treated.Such dosage may be ascertained readily by a person skilled in the art.This dosage regimen may be adjusted to provide the optimal therapeuticresponse.

The compounds of the present invention contain one or more asymmetriccenters and can thus occur as racemates and racemic mixtures, singleenantiomers, diastereomeric mixtures and individual diastereomers. Thepresent invention is meant to comprehend nucleoside compounds having theβ-D stereochemical configuration for the five-membered furanose ring asdepicted in the structural formula below, that is, nucleoside compoundsin which the substituents at C-1 and C-4 of the five-membered furanosering have the β-stereochemical configuration (“up” orientation asdenoted by a bold line).

Some of the compounds described herein contain olefinic double bonds,and unless specified otherwise, are meant to include both E and Zgeometric isomers.

Some of the compounds described herein may exist as tautomers such asketo-enol tautomers. The individual tautomers as well as mixturesthereof are encompassed with compounds of structural formula L Anexample of keto-enol tautomers which are intended to be encompassedwithin the compounds of the present invention is illustrated below:

Compounds of structural formula I may be separated into their individualdiastereoisomers by, for example, fractional crystallization from a,suitable solvent, for example methanol or ethyl acetate or a mixturethereof, or via chiral chromatography using an optically activestationary phase.

Alternatively, any stereoisomer of a compound of the structural formulaI may be obtained by stereospecific synthesis using optically purestarting materials or reagents of known configuration.

The stereochemistry of the substituents at the C-2 and C-3 positions ofthe furanose ring of the compounds of the present invention ofstructural formula I is denoted by squiggly lines which signifies thatsubstituents R¹, R², R³ and R⁴ can have either the α (substituent“down”) or β (substituent “up”) configuration independently of oneanother. Notation of stereochemistry by a bold line as at C-1 and C-4 ofthe furanose ring signifies that the substituent has the β-configuration(substituent “up”).

The compounds of the present invention may be administered in the formof a pharmaceutically acceptable salt. The term “pharmaceuticallyacceptable salt” refers to salts prepared from pharmaceuticallyacceptable non-toxic bases or acids including inorganic or organic basesand inorganic or organic acids. Salts of basic compounds encompassedwithin the term “pharmaceutically acceptable salt” refer to non-toxicsalts of the compounds of this invention which are generally prepared byreacting the free base with a suitable organic or inorganic acid.Representative salts of basic compounds of the present inventioninclude, but are not limited to, the following: acetate,benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate,bromide, camsylate, carbonate, chloride, clavulanate, citrate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate,hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide,isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate,mesylate, methylbromide, methylnitrate, methylsulfate, mucate,napsylate, nitrate, N-methylglucaamine ammonium salt, oleate, oxalate,pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate,polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate,tannate, tartrate, teoclate, tosylate, triethiodide and valerate.Furthermore, where the compounds of the invention carry an acidicmoiety, suitable pharmaceutically acceptable salts thereof include, butare not limited to, salts derived from inorganic bases includingaluminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic, mangamous, potassium, sodium, zinc, and the like.Particularly preferred are the ammonium, calcium, magnesium, potassium,and sodium salts. Salts derived from pharmaceutically acceptable organicnon-toxic bases include salts of primary, secondary, and tertiaryamines, cyclic amines, and basic ion-exchange resins, such as arginine,betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylarninoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine, and the like.

Also, in the case of a carboxylic acid (—COOH) group being present inthe compounds of the present invention, pharmaceutically acceptableesters of carboxylic acid derivatives, such as methyl, ethyl, orpivaloyloxymethyl, can be employed. Included are those ester groupsknown in the art for modifying the solubility or hydrolysischaracteristics for use as sustained-release or prodrug formulations.

Preparation of the Nucleoside Compounds and Derivatives of the Invention

The nucleoside compounds and derivatives thereof of the presentinvention can be prepared following synthetic methodologieswell-established in the practice of nucleoside and nucleotide chemistry.Reference is made to the following text for a description of syntheticmethods used in the preparation of the compounds of the presentinvention: “Chemistry of Nucleosides and Nucleotides,” L. B. Townsend,ed., Vols. 1-3, Plenum Press, 1988, which is incorporated by referenceherein in its entirety.

The examples below provide citations to literature publications, whichcontain details for the preparation of final compounds or intermediatesemployed in the preparation of final compounds of the present invention.The nucleoside compounds of the present invention were preparedaccording to procedures detailed in the following examples. The examplesare not intended to be limitations on the scope of the instant inventionin any way, and they should not be so construed. Those skilled in theart of nucleoside and nucleotide synthesis will readily appreciate thatknown variations of the conditions and processes of the followingpreparative procedures can be used to prepare these and other compoundsof the present invention. All temperatures are degrees Celsius unlessotherwise noted.

EXAMPLE 14-Amino-7-[2-C-methyl-3-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(1-12)

Step A:

3,5-Bis-O-(2,4-dichlorobenzyl)-1-O-methyl-α-D-ribofuranose (1-2)

A mixture of2-O-acetyl-3,5-bis-O-(2,4-dichlorobenzyl)-1-O-methyl-α-D-ribofuranose(1-1) [for preparation, see: Helv. Chim. Acta 78: 486 (1995)] (52.4 g,0.10 mol) in methanolic K₂CO₃ (500 mL, saturated at room temperature)was stirred at room temperature for 45 min. and then concentrated underreduced pressure. The oily residue was suspended in CH₂Cl₂ (500 mL),washed with water (300 mL+5×200 mL) and brine (200 mL), dried (Na₂SO₄),filtered, and concentrated to give the title compound (49.0 g) ascolorless oil, which was used without further purification in Step Bbelow.

¹H NMR (DMSO-d₆ ): δ 3.28 (s, 3H, OCH₃), 3.53 (d, 2H, J_(5,4)=4.5 Hz,H-5a, H-5b), 3.72 (dd, 1H, J_(3,4)=3.6 Hz, J_(3,2)=6.6 Hz, H-3), 3.99(ddd, 1H, J_(2,1)=4.5 Hz, J_(2,OH-2)=9.6 Hz, H-2), 4.07 (m, 1H, H-4),4.50 (s, 2H, CH₂Ph), 4.52, 4.60 (2d, 2H, J_(gem)=13.6 Hz, CH₂Ph), 4.54(d, 1H, OH-2), 4.75 (d, 1H, H-1), 7.32-7.45, 7.52-7.57 (2m, 10H, 2Ph).¹³C NMR (DMSO-d₆) δ 55.40, 69.05, 69.74, 71.29, 72.02, 78.41, 81.45,103.44, 127.83, 127.95, 129.05, 129.28, 131.27, 131.30, 133.22, 133.26,133.55, 133.67, 135.45, 135.92.

Step B:

3,5-Bis-O-(2,4-dichlorobenzyl)-1-O-methyl-α-D-erythro-pentofuranos-2-ulose(1-3)

To an ice-cold suspension of Dess-Martin periodinane (50.0 g, 118 mmol)in anhydrous CH₂Cl₂ (350 mL) under argon (Ar) was added a solution ofthe compound from Step A (36.2 g, 75 mmol) in anhydrous CH₂Cl₂ (200 mL)dropwise over 0.5 h. The reaction mixture was stirred at 0° C. for 0.5 hand then at room temperature for 3 days. The mixture was diluted withanhydrous Et₂O (600 mL) and poured into an ice-cold mixture ofNa₂S₂O₃.5H₂O (180 g) in saturated aqueous NaHCO₃ (1400 mL). The layerswere separated, and the organic layer was washed with saturated aqueousNaHCO₃ (600 mL), water (800 mL) and brine (600 mL), dried (MgSO₄),filtered and evaporated to give the title compound (34.2 g) as acolorless, oil, which was used without further purification in Step Cbelow.

¹H NMR (CDCl₃) δ 3.50 (s, 3H, OCH₃), 3.79 (dd, 1H, J_(5a,5b)=11.3 Hz,J_(5a,4)=3.5 Hz, H-5a), 3.94 (dd, 1H, J_(5b,4)=2.3 Hz, H-5b), 4.20 (dd,1H, J_(3,1)=1.3 Hz, J_(3,4)=8.4 Hz, H-3), 4.37 (ddd, 1H, H-4), 4.58,4.69 (2d, 2H, J_(gem)=13.0 Hz, CH₂Ph), 4.87 (d, 1H, H-1), 4.78, 5.03(2d, 2H, J_(gem)=12.5 Hz, CH₂Ph), 7.19-7.26, 7.31-7.42 (2m, 10H, 2Ph).¹³C NMR (DMSO-d₆) δ 55.72, 69.41, 69.81, 69.98, 77.49, 78.00, 98.54,127.99, 128.06, 129.33, 129.38, 131.36, 131.72, 133.61, 133.63, 133.85,133.97, 134.72, 135.32, 208.21.

Step C:

3,5-Bis-O-(2,4-dichlorobenzyl)-2-C-methyl-1-O-methyl-α-D-ribofuranose(1-4)

To a solution of MeMgBr in anhydrous Et₂O (0.48 M, 300 mL) at −55° C.was added dropwise a solution of the compound from Step B (17.40 g, 36.2mmol) in anhydrous Et₂O (125 mL). The reaction mixture was allowed towarm to −30° C. and stirred for 7 h at −30° C. to −15° C., then pouredinto ice-cold water (500 mL) and the mixture vigorously stirred at roomtemperature for 0.5 h. The mixture was filtered through a Celite pad(10×5 cm) which was thoroughly washed with Et₂O. The organic layer wasdried (MgSO₄), filtered and concentrated. The residue was dissolved inhexanes (˜30 mL), applied onto a silica gel column (10×7 cm, prepackedin hexanes) and eluted with hexanes and hexanes/EtOAc (9/1) to give thetitle compound (16.7 g) as a colorless syrup.

¹H NMR (CDCl₃): δ 1.36 (d, 3H, J_(Me,OH)=0.9 Hz, 2C-Me), 3.33 (q, 1H,OH), 3.41 (d, 1H, J_(3,4)=3.3 Hz), 3.46 (s, 3H, OCH₃), 3.66 (d, 2H,J_(5,4)=3.7 Hz, H-5a, H-5b), 4.18 (apparent q, 1H, H-4), 4.52 (s, 1H,H-1), 4.60 (s, 2H, CH₂Ph), 4.63, 4.81 (2d, 2H, J_(gem)=13.2 Hz, CH₂Ph),7.19-7.26, 7.34-7.43 (2m, 10H, 2Ph). ¹³C NMR (CDCl₃): δ 24.88, 55.45,69.95, 70.24, 70.88, 77.06, 82.18, 83.01, 107.63, 127.32, 129.36,130.01, 130.32, 133.68, 133.78, 134.13, 134.18, 134.45, 134.58.

Step D:

4-Chloro-7-[3,5-bis-O-(2,4-dichlorobenzyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(1-5)

To a solution of the compound from Step C (9.42 g, 19 mmol) in anhydrousdichloromethane (285 mL) at 0° C. was added HBr (5.7 M in acetic acid,20 mL, 114 mmol) dropwise. The resulting solution was stirred at 0° C.for 1 h and then at room temperature for 3 h, evaporated in vacuo andco-evaporated with anhydrous toluene (3×40 mL). The oily residue wasdissolved in anhydrous acetonitrile (50 mL) and added to a solution ofsodium salt of 4-chloro-1H-pyrrolo[2,3-d]pyrimidine [for preparation,see J. Chem. Soc., 131 (1960)] in acetonitrile [generated in situ from4-chloro-1H-pyrrolo[2,3-d]pyrimidine (8.76 g, 57 mmol) in anhydrousacetonitrile (1000 mL), and NaH (60% in mineral oil, 2.28 g, 57 mmol),after 4 h of vigorous stirring at room temperature]. The combinedmixture was stirred at room temperature for 24 h, and then evaporated todryness. The residue was suspended in water (250 mL) and extracted withEtOAc (2×500 mL). The combined extracts were washed with brine (300 mL),dried over Na₂SO₄, filtered and evaporated. The crude product waspurified on a silica gel column (10 cm×10 cm) using ethyl acetate/hexane(1:3 and 1:2) as the eluent. Fractions containing the product werecombined and evaporated in vacuo to give the desired product (5.05 g) asa colorless foam.

¹H NMR (CDCl₃): δ 0.93 (s, 3H, CH₃), 3.09 (s, 1H, OH), 3.78 (dd, 1H,J_(5′,5″)=10.9 Hz, J_(5′,4)=2.5 Hz, H-5′), 3.99 (dd, 1H, J_(5″,4)=2.2Hz, H-5″), 4.23-4.34 (m, 2H, H-3′, H-4′), 4.63, 4.70 (2d, 2H,J_(gem)=12.7 Hz, CH₂Ph), 4.71, 4.80 (2d, 2H, J_(gem)=12.1 Hz, CH₂Ph),6.54 (d, 1H, J_(5,6)=3.8 Hz, H-5), 7.23-7.44 (m, 10H, 2Ph). ¹³C NMR(CDCl₃): δ 21.31, 69.10, 70.41, 70.77, 79.56, 80.41, 81.05, 91.11,100.57, 118.21, 127.04, 127.46, 127.57, 129.73, 129.77, 130.57, 130.99,133.51, 133.99, 134.33, 134.38, 134.74, 135.21, 151.07, 151.15 152.47.

Step E:

4-Chloro-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(1-6)

To a solution of the compound from Step D (5.42 g, 8.8 mmol) indichloromethane (175 mL) at −78° C. was added boron trichloride (1M indichloromethane, 88 mL, 88 mmol) dropwise. The mixture was stirred at−78° C. for 2.5 h, then at −30° C. to −20° C. for 3 h. The reaction wasquenched by addition of methanol/dichloromethane (1:1) (90 mL) and theresulting mixture stirred at −15° C. for 30 min., then neutralized withaqueous ammonia at 0° C. and stirred at room temperature for 15 min. Thesolid was filtered and washed with CH₂Cl₂/MeOH (1/1, 250 mL). Thecombined filtrate was evaporated, and the residue was purified by flashchromatography over silica gel using CH₂Cl₂ and CH₂Cl₂:MeOH (99:1, 98:2,95:5 and 90:10) gradient as the eluent to furnish desired compound (1.73g) as a colorless foam, which turned into an amorphous solid aftertreatment with MeCN.

¹H NMR DMSO-d₆) δ 0.64 (s, 3H, CH₃), 3.61-3.71 (m, 1H, H-5′), 3.79-3.88(m, 1H, H-5″), 3.89-4.01 (m, 2H, H-3′, H-4′), 5.15-5.23 (m, 3H, 2′-OH,3′-OH, 5′-OH), 6.24 (s, 1H, H-1′), 6.72 (d, 1H, J_(5,6)=3.8 Hz, H-5),8.13 (d, 1H, H-6), 8.65 (s, 1H, H-2). ¹³C NMR (DMSO-d₆) δ 20.20, 59.95,72.29, 79.37, 83.16, 91.53, 100.17, 117.63, 128.86, 151.13, 151.19,151.45.

Step F:

4-Amino-7-(2-C-methyl-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(1-7)

To the compound from Step E (1.54 g, 5.1 mmol) was added methanolicammonia (saturated at 0° C.; 150 mL). The mixture was heated in astainless steel autoclave at 85° C. for 14 h, then cooled and evaporatedin vacuo. The crude mixture was purified on a silica gel column withCH₂Cl₂/MeOH (9/1) as eluent to give the title compound as a colorlessfoam (0.8 g), which separated as an amorphous solid after treatment withMeCN. The amorphous solid was recrystallized from methanol/acetonitrile;m.p. 222° C.

¹H NMR (DMSO-d₆): δ 0.62 (s, 3H, CH₃), 3.57-3.67 (m, 1H, H-5′),3.75-3.97 (m, 3H, H-5″, H-4′, H-3′), 5.00 (s, 1H, 2′-OH), 5.04 (d, 1H,J_(3′OH,3′)=6.8 Hz, 3′-OH), 5.06 (t, 1H, J_(5′OH,5′,5″)=5.1 Hz, 5′-OH),6.11 (s, 1H, H-1′), 6.54 (d, 1H, J_(5,6)=3.6 Hz, H-5), 6.97 (br s, 2H,NH₂), 7.44 (d, 1H, H-6), 8.02 (s, 1H, H-2). ¹³C NMR (DMSO-d₆): δ 20.26,60.42, 72.72, 79.30, 82.75, 91.20, 100.13, 103.08, 121.96, 150.37,152.33, 158.15.

LC-MS: Found: 279.10 (M-H⁺); calc. for C₁₂H₁₆N₄O₄+H⁺: 279.11.

Step G:

4-Amino-7-[5-O-(tert-butyldimethylsilyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(1-8)

To a solution of the compound from Step F (457 mg, 1.63 mmol) inanhydrous pyridine (3.5 mL) was added tert-butyldimethylsilyl chloride(370 mg, 2.45 mmol). The reaction mixture was stirred at roomtemperature for 24 h. The reaction mixture was then diluted with ethylacetate (40 mL) which was washed with saturated aqueous sodiumbicarbonate solution (20 mL). The organic layer was separated, driedover anhydrous sodium sulfate, filtered, and evaporated to an oil thatwas subjected to chromatography on silica gel eluting with 10% MeOH inCH₂Cl₂. The appropriate fractions were collected, evaporated, and driedunder high vacuum to furnish the title compound as a colorless foam (516mg).

¹H NMR (DMSO-d₆): δ 7.95 (s, 1H), 7.35 (d, 1H, J=3.4Hz), 6.89 (bs, 2H,NH₂), 6.44 (d, 1H, J=3.4Hz), 6.02 (s, 1H), 5.01-4.98 (m, 2H), 3.92-3.70(m, 3H), 3.40-3.25 (m, 1H), 0.82 (s, 9H), 0.54 (s, 3H), 0.00 (s, 6H).

Step H:

4-(p-Methoxyphenyldiphenylmethylamino)-7-[5-O-(tert-butyldimethylsilyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(1-9)

To a solution of the compound from Step G (394 mg, 1.0 mmol) inanhydrous pyridine (5 mL) was added p-methoxyphenylchlorodiphenylmethane(946 mg, 3.06 mmol) and 4-dimethylaminopyridine (DMAP) (123 mg, 1.0mmol). The reaction mixture was stirred at room temperature for 20 h. Itwas then diluted with ethyl acetate (30 mL) and washed with saturatedaqueous sodium bicarbonate solution (3×15 mL) followed by water(2×15mL). The organic layer was dried over anhydrous Na₂SO₄ andconcentrated to an oil. The crude product was purified using columnchromatography on silica gel eluting with 5% MeOH in CH₂Cl₂. Theappropriate fractions were collected and evaporated to give the titlecompound (540 mg).

¹H NMR (DMSO-d₆): δ 7.85 (s, 1H), 7.65 (s, 1H), 7.41 (d, 1H, J=3.8Hz),7.25-7.03 (m, 12H), 6.78 (d, 1H, J=3.6 Hz), 6.69 (d, 2H, J=9 Hz), 5.97(s, 1H), 5.00-4.94 (m, 21), 3.85-3.62 (m, 4H), 3.59 (s, 3H), 0.83 (s,911), 0.55 (s, 311), 0.003 (s, 6H).

Step I:

4-(p-Methoxyphenyldiphenylmethylamino)-7-[5-O-(tert-butyldimethylsilyl)-3-O-(1-oxo-octyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(1-10)

To a solution of the compound from Step H (400 mg, 0.6 mmol) andanhydrous DMAP (73 mg, 0.6 mmol) in anhydrous CH₂Cl₂ (7 mL) was addedslowly triethylamine (250 μL, 1.8 mmol). To the stirred solution wasadded octanoyl chloride (200 μL, 1.2 mmol) over 15 min. The reactionmixture was stirred for an additional 1.5 h. It was then diluted withmethylene chloride (30 mL) and washed with saturated aqueous sodiumbicarbonate solution (3×10 mL) and water (10 mL). The organic layer wasdried over anhydrous sodium sulfate, filtered, and evaporated. Theresidue was subjected to column chromatography on silica gel elutingwith 5% MeOH in CH₂Cl₂ to afford the title compound as a light yellowfoam (340 mg).

¹H NMR (DMSO-d₆): δ 8.02 (s, 1H), 7.75 (s, 1H), 7.58 (d, 1H, J=3.6 Hz),7.34-7.05 (m, 12H), 7.02 (d, 1H, J=3.6 Hz), 6.79 (d, 2H, J=9.0 Hz), 6.01(s, 1H), 5.61 (s, 1H), 5.34 (d, 1H, J=9.0 Hz), 4.19-4.14 (m, 1H),4.00-3.94 (m, 1H), 3.67-3.62 (m, 4H), 3.48-3.40 (m, 1H), 2.40-2.32 (m,2H), 1.60-1.40 (m, 2H), 1.23 (bs, 8H), 0.91 (s, 9H), 0.84-0.80 (m, 3H),0.67 (s, 3H), 0.07 (s, 6H).

Step J:

4-Amino-7-[5-O-(tert-butyldimethylsilyl)-3-O-(1-oxo-octyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(1-11)

A solution of the compound from Step I (250 mg, 0.31 mmol) in 6:3:1MeOH:acetic acid:H₂O (10 mL) was stirred at 50° C. for 12 h. Thereaction mixture was then concentrated to dryness. The residue wasdiluted with ethyl acetate (30 mL) and washed with saturated aqueoussodium bicarbonate solution (3×15 mL) and water (2×10 mL). The organiclayer was dried over anhydrous sodium sulfate, filtered, and evaporated.The crude product (200 mg) was used without further purification in StepK below. Further purification of a small amount was accomplished bysilica gel column chromatography using 5% MeOH in CH₂Cl₂ as the eluentto give the title compound as a white foam.

¹H NMR (CDCl₃): δ 8.29 (s, 1H), 7.57 (d, 1H, J=3.8 Hz), 6.37 (d, 1H,J=3.8 HZ), 6.28 (s, 1H), 5.33-5.28 (m, 3H), 4.29-4.23 (m, 1), 4.08-4.01(m, 1H), 3.86-3.79 (m, 1H), 2.45-2.37 (m, 2H), 1.69-1.62 (m, 2H),1.29-1.23 (m, 8H), 0.97-0.84 (m, 12H), 0.11 (s, 6H).

Step K:

4-Amino-7-[2-C-methyl-3-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(1-12)

To a solution of the compound from Step J (230 mg, 0.44 mmol) inanhydrous THF (5 mL), was added triethylamine (300 μL, 2.14 mmol) andtriethylamine trihydrofluoride (750 μL, 4.5 mmol). The solution wasstirred overnight at room temperature. The reaction mixture was thendiluted with ethyl acetate (30 mL) and washed with saturated aqueoussodium bicarbonate (3×10 mL) and water (10 mL). After drying the organiclayer over anhydrous sodium sulfate and filtration, the solvent wasevaporated. The resulting oil was purified on a silica gel columneluting with 1:1 acetone/CH₂Cl₂ followed by 10% MeOH in CH₂Cl₂. Theappropriate fractions were concentrated and lyophilized to afford thetitle compound as a colorless powder (90 mg).

¹H NMR (CDCl₃): δ 8.30 (s, 1H), 7.31 (d, 1H, J=3.8 Hz), 6.39 (d, 1H,J=3.8 Hz), 6.16 (s, 1H), 5.44 (d, 1H, J=7.8 Hz), 5.23 (bs, 2H),4.31-4.24 (m, 1H), 4.14-4.06 (m, 1H), 3.84-3.76 (m, 1H), 2.48-2.40 (m,2H), 1.80-1.50 (m, 3H), 1.34-1.23 (m, 7H), 0.95 (s, 31H), 0.88-0.55 (m,3H).

EXAMPLE 24Amino-7-[2-C-methyl-3,5-di-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(2-3)

Step A:

4-(p-Methoxyphenyldiphenylmethylamino)-7-[3-O-(1-oxo-octyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(2-1)

A solution of the compound from Step I of Example 1 (1-10) (300 mg, 0.37mmol), anhydrous triethylamine (300 μL, 2.14 mmol) and triethylaminetrihydrofluoride (750 μL, 4.5 mmol) in anhydrous THP (5 mL) was stirredat room temperature overnight. The reaction mixture was diluted withethyl acetate (50 mL) and washed with saturated aqueous sodiumbicarbonate solution (3×20 mL) followed by water (2×15 mL). The organiclayer was separated, dried over sodium sulfate, filtered, andevaporated. The crude product was purified on a silica gel column using10-15% acetone in CH₂Cl₂ as the eluent. The appropriate fractions werecombined and evaporated to afford the title compound as a colorless foam(240 mg).

¹H NMR (DMSO-d₆): δ 8.03 (s, 1H), 7.79 (s, 1H), 7.56 (d, 1H, J=3.8 Hz),7.38-7.17 (m, 12H), 7.04 (d, 1H, J=3.8 Hz), 6.83 (d, 2H, J=9.0 Hz), 6.13(s, 1H), 5.56 (s, 1H), 5.31 (d, 1H, J=9 Hz), 5.21-5.16 (m, 1H),4.20-4.08 (m, 1H), 3.38-3.70 (m, 41H), 3.65-3.40 (m, 2H), 2.43-2.36 (m,2H), 1.63-1.45 (m, 2H), 1.27 (bs, 8H), 0.91-0.84 (m, 3H), 0.74 (s, 31H).

Step B:

4-(p-Methoxyphenyldiphenylmethylamino)-7-[3,5-di-O-(1-oxo-octyl-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(2-2)

A solution of the compound from Step B (18 mg, 0.026 mmol) and DMAP (3.5mg, 0.028 mmol) in anhydrous CH₂Cl₂ (300 μL) was cooled in an ice bathfor 10 minutes under an argon atmosphere. To this solution was addedtriethylamine (7.5 μL, 0.053 mmol) followed by octanoyl chloride (6.6μL, 0.038 mmol). The reaction mixture was stirred at this temperaturefor 2 h, diluted with CH₂Cl₂ (20 mL) and washed with saturated aqueoussodium bicarbonate solution (2×10 mL) followed by water (10 mL). Thecrude product obtained after evaporation was purified by columnchromatography on silica gel eluting with 10% acetone in CH₂Cl₂. Thetitle compound was obtained as a colorless foam (13.5 mg).

Step C:

4-Amino-7-[2-C-methyl-3,5-di-O-(l-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(2-3)

A solution of the compound from Step B (13 mg, 0.016 mmol) in 6:3:1MeOH:acetic acid:H₂O (500 μL) was stirred at 50° C. for 15 h. Thereaction mixture was then concentrated to dryness. The residue wasdiluted with ethyl acetate (15 mL) and washed with saturated aqueoussodium bicarbonate solution (3×5 mL) and water (2×5 mL). The organiclayer was dried over anhydrous sodium sulfate, filtered, and evaporated.The crude product was purified by silica gel column chromatographyeluting with 10% acetone in dichloromethane to afford the title compoundas a white foam (6.0 mg).

¹H NMR (CDCl₃): δ 8.29 (s, 1H), 7.25 (d, 1H, J=3.4 Hz), 6.40 (d, 1H,J=4.0 Hz), 6.23 (s, 1H), 5.22-5.39 (m, 3H), 4.60-4.39 (m, 4H), 2.47-2.35(m, 4H), 1.82-1.60 (m, 4H), 1.27 (bs, 16 H), 0.87 (s, 3H), 0.873-0.80(m, 6H).

EXAMPLE 34-Amino-7-[2-C-methyl-5-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(3-3)

Step A:

4-(p-Methoxyphenyldiphenylmethylamino)-7-[2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(3-1)

To a solution of the compound 1-9 from Step H of Example 1 in anhydrousTHF, triethylamine (5 eq) and triethylamine trihydrofluoride (10 eq) areadded. The solution is stirred overnight at room temperature. Thereaction mixture is then diluted with ethyl acetate and washed withsaturated aqueous sodium bicarbonate (3×10 mL) followed by water. Afterdrying the organic layer over anhydrous sodium sulfate and filtration,the solvent is removed by evaporation. The resulting oil is purified ona silica gel column eluting with a mixture of dichloromethane andmethanol. The appropriate fractions are concentrated and dried to affordthe title compound as a colorless powder.

Step B:

4-(p-Methoxyphenyldiphenylmethylamino)-7-[2-C-methyl-5-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(3-2)

To a solution of the compound from Step A and DMAP (1.0 eq) in anhydrousCH₂Cl₂ is added slowly triethylamine (2 eq). To the stirred solutionoctanoyl chloride (1.1 eq.) is added over 15 min. The reaction mixtureis stirred for an additional 1.5 h. It is then diluted with methylenechloride and washed with saturated aqueous sodium bicarbonate solutionand water. The organic layer is dried over anhydrous sodium sulfate,filtered, and evaporated. The residue is subjected to columnchromatography on silica gel eluting with a mixture of MeOH in CH₂Cl₂ toafford the title compound.

Step C:

4-Amino-7-[2-C-methyl-5-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(3-3)

A solution of the compound from Step B in 6:3:1 MeOH:acetic acid:H₂O isstirred at 50° C. for 15 h. The reaction mixture is then concentrated todryness. The residue is diluted with ethyl acetate and washed withsaturated aqueous sodium bicarbonate solution and water. The organiclayer is dried over anhydrous sodium sulfate, filtered, and evaporated.The crude product is purified by silica gel column chromatography usinga mixture of acetone and dichloromethane as the eluent to afford thetitle compound.

EXAMPLE 44-Amino-7-[2,3,5-tri-O-(1-oxo-octyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(42)

Step A:

4-(p-Methoxyphenyldiphenylmethylamino)-7-[2,3,5-tri-O-(1-oxo-octyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(4-1)

To a solution of compound 2-2 from Step B of example 2 and DMAP (1.0 eq)in anhydrous CH₂Cl₂ is added slowly triethylamine (2 eq). To the stirredsolution octanoyl chloride (1.1 eq) is added over 15 min. The reactionmixture is stirred for an additional 4 h. It is then diluted withmethylene chloride and washed with saturated aqueous sodium bicarbonatesolution and water. The organic layer is dried over anhydrous sodiumsulfate, filtered, and evaporated. The residue is subjected to columnchromatography on silica gel eluting with mixture of of MeOH in CH₂Cl₂to afford the title compound.

Step B:

4-Amino-7-[2,3,5-tri-O-(1-oxo-octyl)-2-C-methyl-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine(4-2)

A solution of the compound from Step A in 6:3:1 MeOH:acetic acid:H₂O isstirred at 50° C. for 15 h. The reaction mixture is then concentrated todryness. The residue is diluted with ethyl acetate and washed withsaturated aqueous sodium bicarbonate solution and water. The organiclayer is dried over anhydrous sodium sulfate, filtered, and evaporated.The crude product is purified by silica gel column chromatography usinga mixture of acetone and dichloromethane as the eluent to afford thetitle compound.

Biological Assays

The assays employed to measure the inhibition of HCV NS5B polymerase andHCV replication are described below.

The effectiveness of the compounds of the present invention asinhibitors of HCV NS5B RNA-dependent RNA polymerase (RdRp) was measuredin the following assay.

A. Assay for Inhibition of HCV NS5B Polymerase:

This assay was used to measure the ability of the nucleoside derivativesof the present invention to inhibit the enzymatic activity of theRNA-dependent RNA polymerase (NS5B) of the hepatitis C virus (HCV) on aheteromeric RNA template.

Procedure:

Assay Buffer Conditions: (50 μL-total/reaction)

20 mM Tris, pH 7.5

50 μM EDTA

5 mM DTT

2 mM MgCl₂

80 mM KCl

0.4 U/μL RNAsin (Promega, stock is 40 units/μL)

0.75 μg t500 (a 500-nt RNA made using T7 runoff transcription with asequence from the NS2/3 region of the hepatitis C genome)

1.6 μg purified hepatitis C NS5B (form with 21 amino acids C-terminallytruncated)

1 μM A,C,U,GTP (Nucleoside triphosphate mix)

[alpha-³²P]-GTP or [alpha-³³P]-GTP

The compounds were tested at various concentrations up to 100 μM finalconcentration.

An appropriate volume of reaction buffer was made including enzyme andtemplate t500. Nucleoside derivatives of the present invention werepipetted into the wells of a 96-well plate. A mixture of nucleosidetriphosphates (NTP's), including the radiolabeled GTP, was made andpipetted into the wells of a 96-well plate. The reaction was initiatedby addition of the enzyme-template reaction solution and allowed toproceed at room temperature for 1-2 h.

The reaction was quenched by addition of 20 μL 0.5M EDTA, pH 8.0. Blankreactions in which the quench solution was added to the NTPs prior tothe addition of the reaction buffer were included.

50 μL of the quenched reaction were spotted onto DE81 filter disks(Whatman) and allowed to dry for 30 min. The filters were washed with0.3 M ammonium formate, pH 8 (150 ml/wash until the cpm in 1 mL wash isless than 100, usually 6 washes). The filters were counted in 5-mLscintillation fluid in a scintillation counter.

The percentage of inhibition was calculated according to the followingequation:% Inhibition=[1−(cpm in test reaction−cpm in blank)/(cpm in controlreaction−cpm in blank)]×100.

Representative compounds tested in the HCV NS5B polymerase assayexhibited IC₅₀'s less than 100 micromolar.

B. Assay for Inhibition of HCV RNA Replication:

The compounds of the present invention were also evaluated for theirability to affect the replication of Hepatitis C Virus RNA in culturedhepatoma (HuH-7) cells containing a subgenomic HCV Replicon. The detailsof the assay are described below. This Replicon assay is a modificationof that described in V. Lohmann, P. Korner, J -O. Koch, U. Herian, L.Theilmann, and R. Bartenschlager, “Replication of a Sub-genomicHepatitis C Virus RNAs in a Hepatoma Cell Line,” Science 285:110 (1999).

Protocol:

The assay was an in situ Ribonuclease protection, ScintillationProximity based-plate assay (SPA). 10,000-40,000 cells were plated in100-200 μL of media containing 0.8 mg/mL G418 in 96-well cytostar plates(Amersham). Compounds were added to cells at various concentrations upto 100 μM in 1% DMSO at time 0 to 18 h and then cultured for 24-96 h.Cells were fixed (20 min, 10% formalin), permeabilized (20 min, 0.25%Triton X-100/PBS) and hybridized (overnight, 50° C.) with asingle-stranded ³³P RNA probe complementary to the (+) strand NS5B (orother genes) contained in the RNA viral genome. Cells were washed,treated with RNAse, washed, heated to 65° C. and counted in a Top-Count.Inhibition of replication was read as a decrease in counts per minute(cpm).

Human HuH-7 hepatoma cells, which were selected to contain a subgenomicreplicon, carry a cytoplasmic RNA consisting of an HCV 5′ non-translatedregion (NTR), a neomycin selectable marker, an EMCV IRES (internalribosome entry site), and HCV non-structural proteins NS3 through NS5B,followed by the 3′ NTR.

Representative compounds tested in the replication assay exhibitedEC₅₀'s less than 100 micromolar.

The nucleoside derivatives of the present invention were also evaluatedfor cellular toxicity and anti-viral specificity in the counterscreensdescribed below.

C. Counterscreens:

The ability of the nucleoside derivatives of the present invention toinhibit human DNA polymerases was measured in the following assays.

a. Inhibition of Human DNA Polymerases Alpha and Beta:

Reaction Conditions:

50 μL reaction volume

Reaction Buffer Components:

20 mM Tris-HCl, pH 7.5

200 μg/mL bovine serum albumin

100 mM KCl

2 mM β-mercaptoethanol

10 MM MgCl₂

1.6 μM dA, dG, dC, dTTP

α-³³P-dATP

Enzyme and Template:

0.05 mg/mL gapped fish sperm DNA template

0.01 U/μL DNA polymerase α or β

Preparation of Gapped Fish Sperm DNA Template:

Add 5 μL 1M MgCl₂ to 500 μL activated fish sperm DNA (USB 70076);

Warm to 37° C. and add 30 μL of 65 U/μL of exonuclease III (GibcoBRL18013-011);

Incubate 5 min at 37° C.;

Terminate reaction by heating to 65° C. for 10 min;

Load 50-100 μL aliquots onto Bio-spin 6 chromatography columns (Bio-Rad732-6002) equilibrated with 20 mM Tris-HCl, pH 7.5;

Elute by centrifugation at 1,000×g for 4 min;

Pool eluate and measure absorbance at 260 nm to determine concentration.

The DNA template was diluted into an appropriate volume of 20 mMTris-HCl, pH 7.5 and the enzyme was diluted into an appropriate volumeof 20 mM Tris-HCl, containing 2 mM β-mercaptoethanol, and 100 mM KCl.Template and enzyme were pipetted into microcentrifuge tubes or a 96well plate. Blank reactions excluding enzyme and control reactionsexcluding test compound were also prepared using enzyme dilution bufferand test compound solvent, respectively. The reaction was initiated withreaction buffer with components as listed above. The reaction wasincubated for 1 hour at 37° C. The reaction was quenched by the additionof 20 μL 0.5M EDTA. 50 μL of the quenched reaction was spotted ontoWhatman DE81 filter disks and air dried. The filter disks wererepeatedly washed with 150 mL 0.3M ammonium formate, pH 8 until 1 mL ofwash is <100 cpm. The disks were washed twice with 150 mL absoluteethanol and once with 150 mL anhydrous ether, dried and counted in 5 mLscintillation fluid.

The percentage of inhibition was calculated according to the followingequation:% inhibition=[1−(cpm in test reaction−cpm in blank)/(cpm in controlreaction−cpm in blank)]×100.b. Inhibition of Human DNA Polymerase Gamma:

The potential for inhibition of human DNA polymerase gamma was measuredin reactions that included 0.5 ng/μL enzyme; 10 μM dATP, dGTP, dCTP, andTTP; 2 μCi/reaction [α-³³P]-dATP, and 0.4 μg/μL activated fish sperm DNA(purchased from US Biochemical) in a buffer containing 20 mM Tris pH8, 2mM β-mercaptoethanol, 50 mM KCl, 10 mM MgCl₂, and 0.1 μg/μL BSA.Reactions were allowed to proceed for 1 h at 37° C. and were quenched byaddition of 0.5 M EDTA to a final concentration of 142 mM. Productformation was quantified by anion exchange filter binding andscintillation counting. Compounds were tested at up to 50 μM.

The percentage of inhibition was calculated according to the followingequation:% inhibition=[1−(cpm in test reaction−cpm in blank)/(cpm in controlreaction−cpm in blank)]×100.

The ability of the nucleoside derivatives of the present invention toinhibit IV infectivity and HIV spread was measured in the followingassays.

c. HIV Infectivity Assay

Assays were performed with a variant of HeLa Magi cells expressing bothCXCR4 and CCR5 selected for low background β-galactosidase (β-gal)expression. Cells were infected for 48 h, and β-gal production from theintegrated HIV-1 LTR promoter was quantified with a chemiluminescentsubstrate (Galactolight Plus, Tropix, Bedford, Mass.). Inhibitors weretitrated (in duplicate) in twofold serial dilutions starting at 100 μM;percent inhibition at each concentration was calculated in relation tothe control infection.

d. Inhibition of HIV Spread

The ability of the compounds of the present invention to inhibit thespread of the human immunedeficiency virus (HIV) was measured by themethod described in U.S. Pat. No. 5,413,999 (May 9, 1995), and J. P.Vacca, et al., Proc. Natl. Acad. Sci., 91: 4096-4100 (1994), which areincorporated by reference herein in their entirety.

The nucleoside derivatives of the present invention were also screenedfor cytotoxicity against cultured hepatoma (HuH-7) cells containing asubgenomic HCV Replicon in an MTS cell-based assay as described in theassay below. The HuH-7 cell line is described in H. Nakabayashi, et al.,Cancer Res., 42: 3858 (1982).

e. Cytotoxicity Assay:

Cell cultures were prepared in appropriate media at concentrations ofapproximately 1.5×10⁵ cells/mL for suspension cultures in 3 dayincubations and 5.0×10⁴ cells/mL for adherent cultures in 3 dayincubations. 99 μL of cell culture was transferred to wells of a 96-welltissue culture treated plate, and 1 μL of 100-times final concentrationof the test compound in DMSO was added. The plates were incubated at 37°C. and 5% CO₂ for a specified period of time. After the incubationperiod, 20 μL of CellTiter 96 Aqueous One Solution Cell ProliferationAssay reagent (MTS) (Promega) was added to each well and the plates wereincubated at 37° C. and 5% CO₂ for an additional period of time up to 3h. The plates were agitated to mix, well and absorbance at 490 nm wasread using a plate reader. A standard curve of suspension culture cellswas prepared with known cell numbers just prior to the addition of MTSreagent. Metabolically active cells reduce MTS to formazan. Formazanabsorbs at 490 mm. The absorbance at 490 nm in the presence of compoundwas compared to absorbance in cells without any compound added.Reference: Cory, A. H. et al., “Use of an aqueous solubletetrazolium/formazan assay for cell growth assays in culture,” CancerCommun. 3: 207 (1991).

The following assays were employed to measure the activity of thecompounds of the present invention against other RNA-dependent RNAviruses:

a. Determination of In Vitro Antiviral Activity of Compounds AgainstRhinovirus (Cytopathic Effect Inhibition Assay):

Assay conditions are described in the article by Sidwell and Huffman,“Use of disposable microtissue culture plates for antiviral andinterferon induction studies,” Apl. Microbiol. 22: 797-801(1971).

Viruses:

Rhinovirus type 2 (RV-2), strain HGP, was used with KB cells and media(0.1% NaHCO₃, no antibiotics) as stated in the Sidwell and Huffmanreference. The virus, obtained from the ATCC, was from a throat swab ofan adult male with a mild acute febrile upper respiratory illness.

Rhinovirus type 9 (RV-9), strain 211, and rhinovirus type 14 (RV-14),strain Tow, were also obtained from the American Type Culture Collection(ATCC) in Rockville, Md. RV-9 was from human throat washings and RV-14was from a throat swab of a young adult with upper respiratory illness.Both of these viruses were used with HeLa Ohio-1 cells (Dr. Fred Hayden,Univ. of VA) which were human cervical epitheloid carcinoma cells. MEM(Eagle's minimum essential medium) with 5% Fetal Bovine serum (FBS) and0.1% NaHCO₃ was used as the growth medium.

Antiviral test medium for all three virus types was MEM with 5% PBS,0.1% NaHCO₃, 50 μg gentamicin/mL, and 10 mM MgCl₂.

2000 μg/mL was the highest concentration used to assay the compounds ofthe present invention. Virus was added to the assay plate approximately5 min after the test compound. Proper controls were also run. Assayplates were incubated with humidified air and 5% CO₂ at 37° C.Cytotoxicity was monitored in the control cells microscopically formorphologic changes. Regression analysis of the virus CPE data and thetoxicity control data gave the ED50 (50% effective dose) and CC50 (50%cytotoxic concentration). The selectivity index (SI) was calculated bythe formula:SI=CC50÷ED50.b. Determination of In Vitro Antiviral Activity of Compounds AgainstDenpue, Banzi, and Yellow Fever (CPE Inhibition Assay)

Assay details are provided in the Sidwell and Huffman reference above.

Viruses:

Dengue virus type 2, New Guinea strain, was obtained from the Center forDisease Control. Two lines of African green monkey kidney cells wereused to culture the virus (Vero) and to perform antiviral testing(MA-104). Both Yellow fever virus, 17D strain, prepared from infectedmouse brain, and Banzi virus, H 336 strain, isolated from the serum of afebrile boy in South Africa, were obtained from ATCC. Vero cells wereused with both of these viruses and for assay.

Cells and Media:

MA-104 cells (BioWhittaker, Inc., Walkersville, Md.) and Vero cells(ATCC) were used in Medium 199 with 5% PBS and 0.1% NaHCO₃ and withoutantibiotics. Assay medium for dengue, yellow fever, and Banzi viruseswas MEM, 2% FBS, 0.18% NaHCO₃ and 50 μg gentamicin/mL.

Antiviral testing of the compounds of the present invention wasperformed according to the Sidwell and Huffman reference and similar tothe above rhinovirus antiviral testing. Adequate cytopathic effect (CPE)readings were achieved after 5-6 days for each of these viruses.

c. Determination of In Vitro Antiviral Activity of Compounds AgainstWest Nile Virus (CPE Inhibition Assay)

Assay details are provided in the Sidwell and Huffman reference citedabove. West Nile virus, New York isolate derived from crow brain, wasobtained from the Center for Disease Control. Vero cells were grown andused as described above. Test medium was MEM, 1% FBS, 0.1% NaHCO₃ and 50μg gentamicin/mL.

Antiviral testing of the compounds of the present invention wasperformed following the methods of Sidwell and Huffman which are similarto those used to assay for rhinovirus activity. Adequate cytopathiceffect (CPE) readings were achieved after 5-6 days.

d. Determination of In Vitro Antiviral Activity of Compounds AgainstRhino, Yellow Fever, Dengue, Banzi, and West Nile Viruses (Neutral RedUptake Assay)

After performing the CPE inhibition assays above, an additionalcytopathic detection method was used which is described in “MicrotiterAssay for Interferon: Microspectrophotometric Quantitation of CytopathicEffect,” Appl. Environ. Microbiol. 31: 35-38 (1976). A Model EL309microplate reader (Bio-Tek Instruments Inc.) was used to read the assayplate. ED50's and CD50's were calculated as above.

EXAMPLE OF A PHARMACEUTICAL FORMULATION

As a specific embodiment of an oral composition of a compound of thepresent invention, 50 mg of the compound of Example 1 is formulated withsufficient finely divided lactose to provide a total amount of 580 to590 mg to fill a size O hard gelatin capsule.

While the invention has been described and illustrated in reference tospecific embodiments thereof, those skilled in the art will appreciatethat various changes, modifications, and substitutions can be madetherein without departing from the spirit and scope of the invention.For example, effective dosages other than the preferred doses as setforth hereinabove may be applicable as a consequence of variations inthe responsiveness of the human being treated for severity of the HCV,infection. Likewise, the pharmacologic response observed may varyaccording to and depending upon the particular active compound selectedor whether there are present pharmaceutical carriers, as well as thetype of formulation and mode of administration employed, and suchexpected variations or differences in the results are contemplated inaccordance with the objects and practices of the present invention. Itis intended therefore that the invention be limited only by the scope ofthe claims which follow and that such claims be interpreted as broadlyas is reasonable.

1. A compound of structural formula I:

or a pharmaceutically acceptable salt thereof; wherein R¹ is C₁₋₄ alkyl,wherein alkyl is unsubstituted or substituted with hydroxy, amino, C₁₋₄alkoxy, C₁₋₄ alkylthio, or one to three fluorine atoms; R² is amino,fluorine, hydroxy, C₁₋₁₀ alkylcarbonyloxy, mercapto, or C₁₋₄ alkoxy; R³and R⁴ are each independently hydrogen, C₁₋₁₆ alkylcarbonyl, C₂₋₁₈alkenylcarbonyl, C₁₋₁₀ alkyloxycarbonyl, C₃₋₆ cycloalkylcarbonyl, C₃₋₆cycloalkyloxycarbonyl, CH₂O(C═O)C₁₋₄ alkyl, CH(C₁₋₄ alkyl)O(C═O)C₁₋₄alkyl, or an amino acyl residue of structural formula

with the proviso that at least one of R³ and R⁴ is not hydrogen; R⁵ andR⁶ are each independently hydrogen, methyl, hydroxymethyl, orfluoromethyl; R⁷ is hydrogen, C₁₋₄ alkyl, C₂₋₄ alkynyl, halogen, cyano,carboxy, C₁₋₄ alkyloxycarbonyl, azido, amino, C₁₋₄ alkylamino, di(C₁₋₄alkyl)amino, hydroxy, C₁₋₆ alkoxy, C₁₋₆ alkylthio, C₁₋₆ alkylsulfonyl,or (C₁₋₄ alkyl)₀₋₂ aminomethyl; R⁸ is hydrogen, cyano, nitro, C₁₋₃alkyl, NHCONH₂, CONR¹¹R¹¹, CSNR¹¹R¹¹, COOR¹¹, C(═NH)NH₂, hydroxy, C₁₋₃alkoxy, amino, C₁₋₄ alkylamino, di(C₁₋₄ alkyl)amino, halogen,(1,3-oxazol-2-yl), (1,3-thiazol-2-yl), or (imidazol-2-yl); wherein alkylis unsubstituted or substituted with one to three groups independentlyselected from halogen, amino, hydroxy, carboxy, and C₁₋₃ alkoxy; R⁹ ishydrogen, hydroxy, mercapto, halogen, C₁₋₄ alkoxy, C₁₋₄ alkylthio, C₁₋₈alkylcarbonyloxy, C₃₋₆ cycloalkylcarbonyloxy, C₁₋₈ alkyloxycarbonyloxy,C₃₋₆ cycloalkyloxycarbonyloxy, OCH₂CH₂SC(═O)C₁₋₄ alkyl, OCH₂O(C═O)C₁₋₄alkyl, OCH(C₁₋₄ alkyl)O(C═O)C₁₋₄ alkyl, amino, C₁₋₄ alkylamino, di(C₁₋₄alkyl)amino, C₃₋₆ cycloalkylamino, or di(C₃₋₆ cycloalkyl)amino; R¹⁰ ishydrogen, hydroxy, halogen, C₁₋₄ alkoxy, amino, C₁₋₄ alkylamino, di(C₁₋₄alkyl)amino, C₃₋₆ cycloalkylamino, or di(C₃₋₆ cycloalkylamino); each R¹¹is independently hydrogen or C₁₋₆ alkyl; R¹² is hydrogen, C₁₋₄ alkyl, orphenyl C₀₋₂ alkyl; and R¹³ is hydrogen, C₁₋₄ alkyl, C₁₋₄ acyl, benzoyl,C₁₋₄ alkyloxycarbonyl, phenyl C₀₋₂ alkyloxycarbonyl, C₁₋₄alkylaminocarbonyl, phenyl C₀₋₂ alkylaminocarbonyl, C₁₋₄ alkylsulfonyl,or phenyl C₀₋₂ alkylsulfonyl.
 2. The compound of claim 1 of structuralformula II:

or a pharmaceutically acceptable salt thereof; wherein R¹ is C₁₋₃ alkyl,wherein alkyl is unsubstituted or substituted with hydroxy, amino, C₁₋₃alkoxy, C₁₋₃ alkylthio, or one to three fluorine atoms; R² is hydroxy,amino, fluoro, or C₁₋₃ alkoxy; R³ and R⁴ are each independentlyhydrogen, C₁₋₈ alkylcarbonyl, or C₃₋₆ cycloalkylcarbonyl, with theproviso that at least one of R³ and R⁴ is not hydrogen; R⁷ is hydrogen,amino, or C₁₋₄ alkylamino; R⁸ is hydrogen, cyano, methyl, halogen, orCONH₂; and R⁹ and R¹⁰ are each independently hydrogen, halogen, hydroxy,or amino.
 3. The compound of claim 2 wherein R¹ is methyl, fluoromethyl,hydroxymethyl, difluoromethyl, trifluoromethyl, or aminomethyl; R² ishydroxy, amino, fluoro, or methoxy; R³ and R⁴ are each independentlyhydrogen or C₁₋₈ alkylcarbonyl, with the proviso that at least one of R³and R⁴ is not hydrogen; R⁷ is hydrogen or amino; R⁸ is hydrogen, cyano,methyl, halogen, or CONH₂; and R⁹ and R¹⁰ are each independentlyhydrogen, fluoro, hydroxy, or amino.
 4. The compound of claim 1 selectedfrom the group consisting of:4-amino-7-[2-C-methyl-3,5-di-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine;4-amino-7-[2-C-methyl-3-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine;4-amino-7-[2-C-methyl-5-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine; and4-amino-7-[2-C-methyl-2,3,5-tri-O-(1-oxo-octyl)-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidine;or a pharmaceutically acceptable salt thereof.
 5. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 6. The pharmaceutical composition of claim 5 usefulfor inhibiting RNA-dependent RNA viral polymerase, inhibitingRNA-dependent RNA replication, and/or treating RNA-dependent RNA viralinfection.
 7. The pharmaceutical composition of claim 6 wherein saidRNA-dependent RNA viral polymerase is HCV NS5B polymerase, saidRNA-dependent RNA viral replication is HCV replication, and saidRNA-dependent RNA viral infection is HCV infection.
 8. A method ofinhibiting RNA-dependent RNA viral polymerase and/or inhibitingRNA-dependent RNA viral replication comprising administering to a mammalin need of such inhibition an effective amount of a compound accordingto claim
 1. 9. The method of claim 8 wherein said RNA-dependent RNAviral polymerase is HCV NS5B polymerase and said RNA-dependent RNA viralreplication is HCV viral replication.
 10. A method of treatingRNA-dependent RNA viral infection comprising administering to a mammalin need of such treatment an effective amount of a compound according toclaim
 1. 11. The method of claim 10 wherein said RNA-dependent RNA viralinfection is HCV infection.
 12. The method of claim 11 in combinationwith a therapeutically effective amount of another agent active againstHCV.
 13. The method of claim 12 wherein said agent active against HCV isa 2′-C-Me-ribonucleoside; ribavirin; levovirin; thymosin alpha-1;interferon-β; an inhibitor of NS3 serine protease; an inhibitor ofinosine monophosphate dehydrogenase; interferon-α or pegylatedinterferon-α, alone or in combination with ribavirin or levovirin. 14.The method of claim 13 wherein said agent active against HCV isinterferon-α or pegylated interferon-α, alone or in combination withribavirin. 15-20. (canceled)